Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
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DOI：
10.1371/journal.pone.0073849
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发表时间：
2013-09-02
期刊：
影响因子：
3.7
通讯作者：
Gama, Lucio
中科院分区：
文献类型：
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作者：
Russell, Julia N.;Clements, Janice E.;Gama, Lucio
荧光激活细胞分选 (FACS) 是一种敏感且有价值的表征细胞亚群的技术，并且使用这种方法已经取得了巨大的进步。在分选过程之前，通常用甲醛固定细胞，以保留细胞形态并维持表面分子的表达，并确保感染细胞分选的安全性。人们普遍认为，甲醛固定会改变 RNA 和 DNA 的结构和完整性，因此分析这些细胞中的基因表达一直很困难。因此，我们研究了甲醛固定对细胞系、原代白细胞以及从 SIV 感染的猪尾猕猴中分离的细胞中核酸的稳定性和定量的影响。我们开发了一种从固定细胞中提取 RNA 的方法，该方法产生的 RNA 量与我们从新鲜细胞中分离 RNA 的常用方法相同。固定细胞中 RT-qPCR 的 RNA 定量并不总是与未固定细胞中的具有可比性。相比之下，当通过基于探针的 NanoString 系统测量 RNA 时，RNA 定量没有显着差异。此外，我们还证明，当通过单拷贝细胞基因标准化时，通过 qPCR 对固定细胞中的原病毒 DNA 进行定量与对未固定细胞中的原病毒 DNA 进行定量相当。这些结果提供了定量甲醛固定并通过 FACS 分选的细胞中基因表达的系统程序。
Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.